Ligation results: FAILED (sort of)

Below you will see the gel of the ligation reaction. It almost worked. I increased the contrast of the image to identify the successful part of the reaction.

The left most and right most lanes are 1kb ladders, and the 4 middle lanes are the ligation reactions. The 1kb ladder weights are (from the top): 10kb, 8, 6, 5, 4, 3, …

At 6.5kb there should be a band, and there is a feint one. There is another band just below that and I have no idea what that could be. The brightest bands are at 4kb (pALS) and 2.5kb (gel extracted pBR322). There is also another band just below that which I suppose is the other fragment of the pBR322 digestion, but I’m not too sure.

Anyways here is the image:

20130124-164649.jpg

  • Bill

    Do you have a link to a complete ligation schema somewhere? It’s a bit hard to follow this post-by-post. I’m wondering what the small fragments at the bottom of the gel are.

    Also, is this blog your complete notes, or is there a wiki or something I should be looking at to see everything? If not, I suggest you label your gels better — several posts make no mention of what the standards are, for starters. You might remember — for now — what ladder you’re using, but I don’t know and you won’t either in a month. If you can’t be arsed to write it in, you can scan a copy of the product insert or a labeled gel and link to that from every gel — I like to set up a template for “this is how I record results of type X, eg agarose gels” so that I will always use the same format and include complete information. Otherwise I forget to note things like time, voltage/power settings, buffer etc.

    • http://www.iheartanthony.com Anthony Salvagno

      Everything I’m doing now has been previously posted on OpenWetWare a long time ago. And all that information needs to get ported over here in some concise way, I just don’t have the time right now to do so. Hopefully this weekend I can do so. I’m desperately needing setup posts that I can just link to in every post (like I’ve done with the yeast cultures).

      The ligation is a 3 piece ligation with a 4kb piece, a 2.5kb piece, and a 30bp piece that links the two long pieces together. The ladder I’m using is from
      NEB (I have no idea if the link will work). Let me know if there is anything else specifically that I can clear up. Sorry that it’s all confusing.

      • bill

        Here’s a working link: https://www.neb.com/products/n3232-1-kb-dna-ladder

        Are you sure you have to do this as a 3-way? Those are more troublesome than 2-fragment ligations ime. In the gel above, it looks to me like you might be getting some “offlabel” rejoining of the big pieces, despite non-complementary sticky ends (can happen if you add heaps of ligase, which you seem to be doing)… either that or you have some of the unwanted pBR chunk in there messing things up.

        There are a few things you can do to help a problematic ligation along — molecular crowding (add some PEG), tweak the ligase concentration (less is usually better), I forget what-all else.

        Of course, you could just try running a gel way out for max separation and extracting the apparent 6.5kb species, then drop that into some competent cells and see if they’ll give you what you want. Is there selection on the final plasmid? Even better, is there blue-white selection? If yes, you might as well try to see if you can pull out a clone.